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Brain Imaging Technologies and Their Applications in Neuroscience

By Carolyn Asbury, Ph.D.

With appreciation to John A. Detre, M.D., Ulrich von Andrian, M.D., Ph.D., and

Michael L. Dustin, Ph.D. for their expert guidance

Section I

Introduction

Imaging is becoming an increasingly important tool in both research and

clinical care. A range of imaging technologies now provide unprecedented

sensitivity to visualization of brain structure and function from the level of

individual molecules to the whole brain. Many imaging methods are

noninvasive and allow dynamic processes to be monitored over time. Imaging is

enabling researchers to identify neural networks involved in cognitive

processes; understand disease pathways; recognize and diagnose diseases

early, when they are most effectively treated; and determine how therapies

work.

Moreover, as in other areas of biomedical research, these opportunities

are interactive. As an example, imaging can provide a better understanding

about a disease process that leads to discovery of potential therapies that

intervene in that process. Thereafter, imaging can help provide a better

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understanding about how that drug or therapy works at the molecular level,

leading to a more precise understanding of the disease process and then to

development of a more highly specific drug to treat it.

Different types of imaging are used to reveal brain structure (anatomy),

physiology (functions), and biochemical actions of individual cells and of the

molecules that compose them, and of cells’ functions, behaviors and

interactions. The three main categories, therefore, are often referred to as

structural, functional and molecular imaging. While many imaging techniques

are used throughout the body, the descriptions provided here focus on their use

in the nervous system, primarily the brain.

Alone and in combination, these imaging techniques are transforming

our understanding of how the brain functions, how immune cells function, and

how immune cells interact with the brain in health and disease.

Imaging’s Evolution: Early Structural Imaging Techniques

While many structural and functional imaging techniques are relatively

recent, the origin of structural imaging was the X-ray, developed in 1895. X-rays

measure the density of tissues. X-rays use photons, a quantum of visible light

that possesses energy; the photons are passed through the body and deflected

and absorbed to different degrees by the person’s tissues. They are recorded

as they pass out of the body onto a silver halide film. Dense structures such as

bone, which block most of the photons, appear white; structures containing air

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appear black; and muscle, fat and fluids appear in various shades of gray. This

technology was the clinician’s main imaging tool for more than half of the 20th

century.

A related early technique was angiography, in which a radiopaque dye

was injected through a catheter into a blood vessel to detect a blockage or

narrowing or architecture of downstream vessels. The vessels were outlined on

x-ray as white. Angiography was used to visualize arteries anywhere in the

body, including the neck and brain.

Computerization transformed the x-ray in the 1970s, with the

development of Computer Assisted Tomography (CT), and its two main

developers received the Nobel Prize in Medicine or Physiology in 1979. This

technology uses special x-ray equipment to obtain three-dimensional

anatomical images of bone, soft tissues and air in the entire body, including the

head. An x-ray emitter is rotated around a patient. It measures the rays’

intensities from different angles. For brain imaging, numerous X-ray beams

are passed through the head at different angles. Special sensors measure the

amount of radiation that is absorbed by different tissues. Then, a computer uses

the differences in X-ray absorption to form cross-sectional images or “slices” of

brain called “tomograms.”

CT imaging was the first technique, for instance, to show clear evidence,

during life, of decreases in the amount of brain tissue in older compared to

younger people. CT can be used with or without contrast agents (dyes, such as

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iodine, that make structures easier to see), but use of contrast enables CT to

show bone, soft tissues and blood vessels in the same images.

Because CT can be done quickly, it is especially useful in emergency

trauma situations, showing any abnormalities in brain structure including brain

swelling, or bleeding arising from ruptured aneurysms, hemorrhagic stroke (a

ruptured blood vessel), and head injury.

Ultrasound, another early technique developed in the 1930’s-40’s, was

primarily used neurologically until the 1960s to try to identify brain tumors.

Ultrasound uses sound waves to determine the locations of surfaces within

tissues, and differentiates surfaces from fluids. It does so by measuring the time

that passes between the production of an ultrasonic pulse and the echo created

when the surface reflects the pulse. But, when scientists determined that the

skull significantly distorts the signals, its use for this purpose stopped while its

use in obstetrics and gynecology—to image the fetus in utero and to detect

ovarian tumors—became widespread.

Fortuitously, abandonment of ultrasound to try to detect brain tumors

came at the same time that new radiological technologies for brain imaging

such as magnetic resonance imaging (MRI) were emerging. Beginning in the

1980s, however, new ultrasound techniques (“Laser Doppler Ultrasound”)

began to evolve; these techniques employ laser technology to combine

information from both light and sound and have become a vital part of intensive

monitoring of cerebral blood flow in patients with severe head trauma. These

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technologies and their uses are described later in the section on Electrical and

Doppler Ultrasonic Imaging Techniques.

Entrance of Physiological Imaging and Highly Sensitive Structural

Imaging

Physiological imaging techniques measure changes in cerebral blood

flow (CBF) and brain metabolism. These measurements complement structural

imaging studies by providing information on regional brain function either at rest

or in response to specific perturbations and how it is altered by brain disorders.

Positron Emission Tomography (PET) was the first major technology to

measure physiological functioning in the brain. In PET scanning, the regional

distribution of exogenously administered positron emitting tracers is measured

using tomographic imaging. The first PET tracer to be used in humans was 18F-

deoxyglucose, which distributes according to regional glucose utilization.

Because water is freely diffusible from the blood to the brain, 15O-H2O provides

a PET tracer for measuring cerebral blood flow, and was another early tracer

used for measuring regional brain function.

When introduced clinically in the 1970s, PET provided a fundamentally

new opportunity to explore the parts of the brain that were activated in

undertaking specific tasks, a role it dominated for more than a decade. This

application of PET is predicated on the observation that changes in regional

neural activity are coupled to changes in regional cerebral blood flow and

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metabolism. More recently the main functions for PET are focused on the study

of neurotransmitters (electrochemical signals passed from one brain cell to

another to communicate), the actions of pharmaceutical drugs, and the

expression of specific genes in the brain.

Additionally, in recent years a few PET tracers have been developed that

attach solely to the protein beta amyloid, which builds up in the brains of

patients with mild cognitive impairment (MCI) and Alzheimer’s disease. PET

imaging with these agents has the potential, along with cognitive tests, to

diagnose Alzheimer’s disease in patients, and to identify patterns that may

predict which patients with MCI will develop Alzheimer’s disease.

Due to PET’s ability to measure tiny concentrations of the radioisotope

tracer used, its measurements are exquisitely sensitive. But its utility—

especially clinically—compared to its use in research, is compromised to some

extent by the non-specific nature of the changes in CBF and metabolism and

the need to actually make the radioisotopes at the clinical site.

Radioisotopes, produced by huge and expensive cyclotrons, need to be

made at the site because they decay quickly and so must be used soon after

they are made. This rapid decay is referred to as having a short “half-life,” which

is the time it takes for 50 percent of the radioactivity to decay. Because of the

tiny amount of radioisotope used and its rapid decay, PET tracers generally

produce no adverse effects in patients. The tracer is injected into a patient’s

vein and travels to the brain. The positrons rapidly collide with electrons and

release gamma rays oriented in opposite directions along exactly the same line.

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These gamma rays are detected by two sensors simultaneously, enabling

computers to precisely pinpoint the brain locations of interest.

Due to the need for the expensive cyclotron at the clinical site and the

subsequent development of alternative physiological imaging techniques, PET

is not used extensively to study brain areas that are activated when undertaking

a specific cognitive or motor task (“task activation” studies), Instead, one of its

current major uses is in research on excitatory and inhibitory neurotransmitters

(electrochemical messages passed from one brain cell to another to

communicate). Each neurotransmitter, such as dopamine, GABA, serotonin and

others, attaches to a specialized receptor on a brain cell that receives the

message. In this way, PET identifies brain cell networks using a specific

neurotransmitter to communicate, and also helps to see whether abnormally

high or low transmitter levels are associated with specific brain conditions.

As an example, Parkinson’s disease is characterized by low levels of the

neurotransmitter dopamine. PET is used in Parkinson’s disease research

primarily to assess the effects of experimental treatments on dopamine and to

determine exactly how they work. Also, though not often used for this

purpose, PET imaging of the dopamine network (dopaminergic system) can

help to differentiate early stage Parkinson’s disease from other “parkinsonian”

disorders (multiple system atrophy, progressive supranuclear palsy or

Huntington’s disease).

In the case of PET exploration of dopamine (as for other

neurotransmitters), a radioisotope is attached to molecules that look like

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dopamine and, when introduced intravenously into a person, are taken up by

the same cells that take up dopamine. The radioisotopes make their way to the

brain and concentrate there. PET then images the amount of the labeled

molecules that has been taken up by brain cells to measure dopamine levels.

Because PET tracers bind to many of the same receptors that

pharmaceutical drugs bind to, PET is also widely used to study how

pharmaceuticals act in the brain. Additionally, PET can repeatedly and

quantitatively image gene expression (proteins produced by genes) in the brain,

to provide new insights into the contributions of specific genes to brain

disorders and diseases.

More recently, a few specific PET tracers, such as PIB-PET (Pittsburgh

Compound B”) and “florbetapir -F18 PET” have been developed that bind solely

to beta amyloid proteins, which accumulate in the brains of people with

presymptomatic mild cognitive impairment (MCI), overt MCI, and those with

Alzheimer’s disease, but not in the brains of cognitively healthy adults.

Importantly, not all people with presymptomatic or overt MCI go on to develop

Alzheimer’s disease. Scientists may be able to identify the patterns that indicate

which people with presymtomatic or overt MCI will go on to develop Alzheimer’s

disease and which will not, by imaging all three groups of patients serially over

several years.

Because PIB-PET has an extremely short half-life of only 20 minutes, it

is used for research only at academic centers. A Food and Drug administration

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(FDA) advisory committee in early 2011 recommended that Florbetapir F-18,

which has a relatively longer half-life than PIB-PET, be considered for FDA

approval as the first generally available physiological diagnostic tool for

Alzheimer’s disease, to be used in conjunction with cognitive tests. The

committee advised that approval be contingent upon the manufacturer

developing a training program for clinicians that successfully teaches them how

to consistently read the scans accurately.

An imaging technology that is similar to PET is Single Photon Emission

Computed Tomography (SPECT). It is used for most of the same purposes as

PET, but is less expensive and more convenient for clinical use. SPECT began

to be widely employed clinically in the 1980s because, unlike PET, it detects

directly emitted gamma-rays from commercially available stable radioisotopes.

These isotopes are larger and have a longer half-life than those used in PET,

but the imaging is less precise. So, patients who are seen in clinics other than

at academic research institutions are far more likely to receive SPECT rather

than PET scans. Like PET, however, many of its clinical functions have been

taken over by other physiological imaging techniques, most notably Magnetic

Resonance Imaging (MRI).

Non-invasive Structural and Physiological Imaging: MRI Technologies

Magnetic Resonance Imaging (MRI) is based on the principle of nuclear

magnetic resonance and uses radiofrequency waves to probe tissue structure

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and function without requiring exposure to ionizing radiation. The two

researchers who made MRI clinically feasible in the 1980s by building on initial

discoveries of the 1930s won the Nobel Prize in Physiology or Medicine in

2003.

Clinically, MRI has become the most important diagnostic imaging

modality in neuroscience. One of the many benefits of MRI in the central

nervous system is that the radiofrequency signals readily penetrate the skull

and spinal column, allowing the tissue within it to be images with no

interference. MRI provides the best visualization of parenchymal abnormalities

in the brain and spinal cord including tumors, demyelinating lesions, infections,

vascular lesions such as stroke, developmental abnormalities, and traumatic

injuries.

There are numerous variations on MRI that are also in wide clinical use.

These include flow-sensitive approaches termed magnetic resonance

angiography and magnetic resonance venography that are used to detect

stenosis (narrowing) or clotting of arteries and veins, diffusion sensitized MRI

that can be used to detect acute strokes just minutes after their onset, and

perfusion sensitized MRI that can demonstrate regional cerebral blood flow and

blood volume. In addition to MRI’s uses in clinical care, functional MRI (fMRI) is

used to identify specific brain areas involved in activation of motor and cognitive

tasks, and to detect reorganization in the brain following injury to a localized

area.

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Most MRI techniques use signals from water, which constitutes about

two-thirds of human body weight, to develop information on brain structures and

functions. Protons (positively charged particles) in hydrogen molecules in water

produce the signal when exposed to a strong magnetic field. The MRI machine

contains the magnet. Structural MRI measures the nuclear magnetic resonance

of water protons to create a computerized three-dimensional image of tissues.

More specifically, protons in the nuclei of hydrogen atoms in water move

(oscillate) between two points and vibrate when they are exposed to a strong

magnetic field. They absorb energy in the frequency of radio waves; and then

they remit this energy in the same radiofrequency (a process called resonance)

when they return to their original state. MRI uses the body’s own molecules,

while PET and SPECT require introduction of a radioactive label into the body

with a drawback of exposure to low levels of ionizing radiation.

Small differences in the protons’ oscillations are mathematically analyzed

by computer to build a three-dimensional image of tissues. Variations that occur

in the molecular environment of water located in different brain structures and

compartments provide contrast, and the ability to see the spatial orientation of

various brain structures. The molecular environment of water is also affected by

disease processes.

The contrast differentiates the brain’s gray matter (primarily nerve cell

bodies) from white matter (primarily axons and their myelin sheaths) which are

the nerve cell communication cables that connect brain regions. Structural MRI

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undertaken serially over a two-year period, for instance, shows that the brain’s

hippocampus (primarily gray matter) becomes progressively smaller

(degenerates) in adults with Alzheimer’s compared to adults who are cognitively

healthy.

Functional MRI (fMRI) shows the brain in action; scientists use it

extensively to elucidate processes involved in higher cognitive functioning. It is

highly sensitive so it can detect small changes, and is relatively inexpensive

compared to PET, so it is the method of choice for identifying areas of the brain

that are activated when a person undertakes a specific cognitive or motor task.

It is an indirect measure, however, because the time it takes for dynamic

changes to occur in blood flow is much longer than that for neurons to fire off

their electrochemical messages. Functional MRI can be used to study the

reorganization of function following injury to a single brain area.

Like functional imaging with PET, functional MRI is based on the

principle that changes in regional cerebral blood flow and metabolism are

coupled to changes in regional neural activity involved in brain functioning, such

as memorizing a phrase or remembering a name. Almost all fMRI techniques

use the contrast mechanism called BOLD (blood oxygenation level dependent)

MRI. BOLD contrast reflects a complex interaction between the volume of

blood, its flow, and its transport of oxygen by an iron-containing protein in red

blood cells. Functional contrast is produced only when the oxygen is released

from iron and taken up by brain cells. Loss of the oxygen enables iron to

become highly magnetized when exposed to the MRI magnetic field.

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In addition to using BOLD contrast with fMRI to measure task activation,

a perfusion contrast used with fMRI—called arterial spin labeling (ASL)—can be

used to quantify regional cerebral blood flow noninvasively. Whereas BOLD

fMRI is primarily sensitive to changes in regional brain function, ASL MRI

provides absolute quantification of cerebral blood flow, which renders it

sensitive to both static function and changes occurring over longer intervals.

For example, ASL MRI can detect differences in brain function between

individuals with different genotypes (genetic make-up) or the effects of

chronically administered drugs on regional brain function. However, these

capabilities still rely on a coupling between changes in regional neural activity

and changes in cerebral blood flow, so unlike PET performed with molecular

tracers, ASL-fMRI will not show what the drug is doing at a molecular level once

it gets to its target.

In addition to fMRI, there are several other major MRI techniques. Each

technique has a highly specialized function.

Diffusion-tensor MRI (DTMRI) measures microscopic water motion in any

tissue, and in the brain this motion is facilitated along white matter tracts (the

brain’s communication cables that connect brain regions). Computerized

mathematical models then construct the images of the white matter tracts.

DTMRI, therefore, is used to visualize white matter tracts connecting different

parts of neural networks in the brain. It is used extensively in pre-surgical

planning, such as for removal of a brain tumor, to ensure that these tracts are

spared during surgery. Additionally, DTMRI has been applied to the study of

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neurological conditions, such ADHD and other developmental disorders, that

are thought to arise from problems in white matter connections. Related studies

use PET imaging to explore possible alterations in specific neurotransmitters in

these disorders.

Diffusion-weighted MRI shows whether brain tissue has been damaged

due to insufficient blood flow to the tissue. DWMRI can visualize tissue within

minutes after it is damaged by an “ischemic” injury (such as a stroke-producing

blood clot), to allow early identification of the damage.

Perfusion-weighted MRI can show areas of the brain in which blood flow

has been altered based on the time course of regional signal changes induced

by an exogenously administered MRI contrast agent.

Diffusion-Perfusion-weighted MRI can be used together to estimate the

“ischemic penumbra,” the tissue that has suffered from reduced blood flow but

has not yet died. This tissue is the target of intensive therapy for patients who

have suffered an ischemic stroke.

Magnetic Resonance Spectroscopy (MRS) focuses on magnetic

resonance signals from molecules other than water. Several molecules of

interest can be measured using MRS from 1H or 31P. In general, MRS has

much poorer spatial resolution than MRI, but it has greater specificity. It is a

tool that helps to characterize brain diseases according to the natural history of

the chemical changes that they produce over time. MRS is conducted in an MRI

scanner and like MRI it uses magnetization and radio waves. Instead of

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creating an image, however, MRS produces a spectrum that reflects the

concentrations of various molecules—identified according to their chemical

composition—in a specific area. Each type of molecule has a unique radio wave

frequency (“radiofrequency’). The strength of a molecule’s radiofrequency

depends on how much of the molecule is concentrated in a specific area. While

MRS has much less resolution and sensitivity than PET or SPECT in measuring

biochemical changes, its non-invasive nature makes it highly preferable to use

in studies that contrast biochemical changes in healthy study volunteers from

those in patients with specific brain diseases.

MRS can be used to identify the size and stage of specific kinds of brain

tumors that are known to contain high levels of certain chemicals. Additionally,

beginning in about 2005, MRS has been found able to detect immature

(“progenitor”) cells in the brain that develop into neurons and other cells in the

brain (a process called “neurogenesis”). The ability to track these cells could

lead to important advances in understanding healthy and disordered brain

development in children. This ability to track progenitor cells also may provide

information on whether neurogenesis slows in adulthood and, if so, whether this

lowered rate of brain cell production has serious consequences for adults with

degenerative diseases who cannot replace substantial numbers of dying brain

cells.

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Electrical Recording and Ultrasound Imaging Techniques

Whereas fMRI and PET are based on the coupling of neural and

vascular (blood flow) activities, electrophysiological methods directly reflect

brain cells’ electrical activity. These non-invasive methods include

electroencephalography (EEG) and magnetoencephalography (MEG).

EEG measures the electrical activity that is produced by neurons as

recorded from electrodes placed along the scalp. MEG maps brain activity by

measuring magnetic fields that are generated by neural activity in the brain.

Both EEG and MEG provide information about global as well as regional neural

activity, but with MEG there is less distortion of the electrical signals. Often one

or the other of these electrophysiological methods is combined with fMRI or

PET to provide complementary information about normal and disturbed brain

function.

EEG is used clinically to measure physiological manifestations of

abnormal cortical excitability, primarily in the diagnosis and management of

epilepsy and other seizure disorders. It is also used with other many other

measures in intensive care to monitor head-injured patients in coma, providing

information that helps physicians assess patients’ prognosis. EEG is also used

to study sleep disorders. EEG recordings can be conducted while a patient is

inside the MR scanner. EEG and fMRI are used together, for instance, to

localize where in the brain a seizure starts and where it spreads thereafter.

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MEG, by measuring magnetic fields, is used to investigate the basis of

sensory processing and motor planning in the brain. MEG is used with MRI in

brain tumor patients prior to their surgery to identify the hemisphere controlling

language and to precisely locate the areas involved in expressive and receptive

language so that surgeons can spare these areas during surgery. Sometimes,

patients who will be undergoing this pre-surgical planning will agree to

participate during the MEG/MRI procedure in research designed to explore

brain processes that may be involved in stuttering, or in memory.

Transcranial magnetic stimulation (TMS) is a non-invasive technique that

is used to map cortical functions in the brain, such as identifying motor or

speech areas. With TMS, a large electromagnetic coil is placed on the scalp,

near the forehead. An electromagnet is then used to create a rapidly changing

magnetic field, inducing weak electric currents. It increases plasticity and

excitability of neural circuits.

Unlike TMS, repetitive TMS (rTMS) is used as a therapeutic intervention,

rather than cortical mapping tool. In stroke patients with motor deficits, rTMS is

used to try to restore the balance of excitation between motor cortices in each

brain hemisphere. Changes in signals from the motor cortex can be associated

with improvements in muscle movements, such as raising a finger. Additionally,

as of 2008, rTMS is approved by the Food and Drug Administration for non-

invasive treatment of depression. Researchers continue to gain a better

understanding of mechanisms of actions, and optimal doses (such as frequency

and patterns of delivery). This technique is also being tested experimentally in

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other neurological conditions such Parkinson’s disease, dystonia and

schizophrenia. Like another technique called “deep brain stimulation” (DBS),

TMS functions both to provide information on brain functions and to treat some

functions.

Deep brain stimulation (DBS) involves implanting electrodes in specific

areas in the brain and externally stimulating the electrodes to measure electrical

activities of neurons and their electrochemical pathways. DBS is currently

approved by the FDA for treatment of intractable Parkinson’s disease and

essential tremor. It is also being studied at research centers for treatment of

severe intractable depression, obsessive-compulsive disorder, Tourette’s

syndrome and other conditions.

In addition, however, in a few highly specialized research centers,

neurosurgeons and cognitive scientists are undertaking DBS electrical imaging

to begin to explore the neuronal underpinnings of cognition. The studies are

undertaken in patients with epilepsy, with their consent, while the patients

participate in pre-surgical planning to identify the specific location of the origin

of seizures that cannot be controlled with medication, so that essential areas

can be spared during surgical treatment for these intractable seizures. DBS

electrodes transmit signals from nearby cells when those cells are active in a

specific task, such as naming, responding to a happy or sad face, or involved in

movement, such as raising a finger.

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Laser Doppler Ultrasound is a non-invasive and highly sensitive method

for measuring even tiny changes in the rate of blood flow velocity (speed) within

arteries throughout the body, including the brain. Its primary use in the brain is

for monitoring severely head injured patients, especially those in coma, in

intensive care units. It is used in combination with other measures (such as

EEG, described above). In fact, it is one of many components of multi-modal

monitoring of brain oxygenation and metabolism that help physicians predict a

patient’s prognosis and measure patients’ responses to various therapies. It is

also used to confirm brain death, the irreversible cessation of all functions of the

whole brain.

The technology is based on the principle that sound waves change pitch

when combined with motion, in this case movement of red blood cells. The low

level laser light is able to penetrate thin areas of the skull, enabling intensive

care practitioners to monitor any changes in the ultrasonic signals from patients’

basal cerebral arteries.

Laser Doppler Ultrasound is often used to image the carotid artery to

determine whether a major blockage is present that is decreasing blood supply

to the brain in patients suspected of having a transient ischemic attack or

stroke. In heart disease patients, the technique helps to assess the effects of

angiogenesis treatment (promoting the growth of blood vessels in a damaged

heart); in cancer patients, the technique measures the effects of anti-

angiogenesis treatment (cutting off blood supply to a tumor). Additionally, it is

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used to follow the migration of injected drug therapies that travel to the

abdomen, chest and legs.

Researchers are working to develop ultrasound probes (micro-bubbles

that reflect sound from specific molecular targets) to provide molecular

information. If this work is successful, ultrasound could become a useful

molecular imaging technology.

Cellular and Molecular Imaging

Molecular and cellular imaging techniques answer questions about

normal biochemical activities of cells and their molecules, and how these are

altered by disease, injury and their treatments, but they do so at a much higher

resolution in space and time than do PET, SPECT and MRS. Cellular and

molecular imaging techniques use several types of light microscopes, and

various types of “optical probes,” which are molecules that have been specially

labeled to emit light of various wavelengths, to “contrast” the target cells of

interest from other cells.

Molecular imaging, therefore, exploits specific molecules for image

contrast. This refers to the ability to measure and characterize cellular and

molecular activities in living animals or humans, or in their tissues, by using

contrast to identify and follow actions of only the specifically labeled molecules.

While “intravital” light microscopy—used to visualize live organisms—was

developed more than 170 years ago, the development in recent times of many

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types of highly specialized light-emitting probes, advances in specialized light

microscopes, and computerization have transformed the science of optical

imaging.

There are two general types of intravital cellular imaging technologies:

microscopic and macroscopic. Microscopic techniques are used to image

molecules in tissues from humans and laboratory animals, and in live small

laboratory animals. Macroscopic techniques are used in live small and a few

large laboratory animals.

Intravital microscopic imaging is undertaken in tissue cultures from

humans or animals, with tissues that have been biopsied or surgically removed;

this imaging also can be undertaken in small laboratory animals to visualize the

actions of labeled cells in a specific location. The cells of interest, whether being

viewed in tissue culture or in small laboratory animals, are labeled with one of

several types of light-emitting probes of differing wave lengths and visualized

with one of several types of microscopes; each combination has specialized

advantages.

Intravital macroscopic imaging differs from intravital microscopy in that

macroscopic imaging is undertaken only in living laboratory animals, not in

tissue cultures. Moreover, unlike microscopic imaging in live small animals

where the image is confined to the exact location under view, intravital

macroscopic imaging provides the capacity to visualize labeled light-emitting

cells everywhere they are located within the animal’s body and anywhere these

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cells travel to within the body. Additionally, while macroscopic imaging is usually

undertaken in small laboratory animals, it also can be used in a few large

laboratory animals, such as sheep and pigs.

Both microscopic and macroscopic cellular imaging technologies utilize

many of the same types of light-emitting probes to contrast the cells of interest

from all other cells. Some can only show molecules located near the skin, while

others can show molecules that are deeper within tissues. The types of

microscopes and probes used vary depending upon the scientific questions

being asked and the locations of target cells. For instance, certain combinations

of microscopes and probes are used to visualize a single labeled molecule in a

cell to see what it does, while other combinations show how one molecule

influences another. Opportunities with microscopic and macroscopic imaging

span the range from whole animals, to whole organs, to single cells, and single

molecules.

In fact, there are myriad purposes for using molecular and cellular

imaging techniques, but among some of the most common are the following.

The techniques reveal biochemical activities involved in the: architecture of a

cell; dynamic interactions between two cells or molecules, or among many cells

or molecules; gene expression (the production by a gene of its protein, which

has evolved to carry out a specific function); cell division (proliferation of new

healthy or cancerous cells); cell death; assault and killing of cells by immune

cells or infectious agents; processes of tissue integrity and tissue degeneration;

wound healing; angiogenesis (formation of new blood vessels such as during

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brain development, or to supply brain tissues following a stroke, or to provide

increased blood and its nutrients to fast-growing tumors); gas exchange;

cellular or molecular “trafficking”-such as the migration of immune cells into and

within the brain in response to injury or infection; and to identify where, within a

specific tissue, a particular molecular activity occurs. Imaging of these and other

activities provides information about the normal development of cells and their

biochemical activities, cells’ responses to attack or injury, and how therapies—

such as drugs—alter the cells’ responses.

Optical Imaging Probes

Advances in molecular and cellular imaging are largely due to the

development of major types of light-emitting probes and ingenious ways of

labeling them for use in living laboratory animals and, in a few instances, in

endoscopic imaging in humans. Bioluminescent and fluorescent probes are two

major types.

Bioluminescent probes use “luciferase,” an enzyme, to generate and

emit light by an organism, providing real-time analyses of disease processes at

the molecular level in living organisms, including laboratory animals. Luciferase

is the enzyme in fireflies and glowworms that makes them light up. These are

the best known examples of organisms that naturally produce bioluminescence,

but deep sea marine organisms and some bacteria and fungi also produce

bioluminescence. A bioluminescent probe is prevalently used in studying

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infections and cancer progression. There are many organic fluorescent probes,

such as fluorescein, rhodamine, acridine dyes, phycoerythin and others. There

also are bioluminescent protein probes. In fact, it was during isolation of a

bioluminescent protein that fluorescent probes were discovered.

Fluorescent protein probes are green fluorescent protein, its yellow, blue

and cyan-colored mutants, and red fluorescent proteins. In addition, there are

hundreds of other fluorescent probes that are not fluorescent proteins.

Fluorescent probes are introduced into an animal and visualized in the animal

or its tissue cultures when excited by ultraviolet or visible light and viewed with

optical imaging techniques. Fluorescence is the absorption and subsequent re-

radiation of light by an organism. Fluorescence was known and used for

microscopy, including intravital imaging, for many years prior to the discovery of

fluorescent proteins.

The discovery of fluorescent proteins occurred after scientists, isolating a

blue bioluminescent protein from a specific type of jellyfish, observed another

protein that produced green fluorescence when illuminated with ultraviolet light.

The gene for green fluorescent protein was cloned in the early 1990s; but its

utility as a molecular probe occurred later, after scientists used fusion products

to track gene expression in bacteria and nematodes.

The colored proteins, plus fusion proteins and biosensors—all of which

are referred to as fluorescent proteins—are used primarily to visualize

molecules in living cells. Moreover, multiple (different colored) fluorescent

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probes can simultaneously identify several target molecules within a cell and

show their actions. The fluorescent proteins are fused to specific proteins and

enzymes in the laboratory and primarily introduced into the animal through

production of “transgenic” strains, whereby the fluorescent protein is introduced

into the germline (sequence of germ cells containing genetic material), often

under the control of tissue-specific or cell type-specific promoters. In another

approach, engineered genes that encode fluorescent protein fusions, rather

than the proteins themselves, are introduced into the animals by attaching them

to harmless viruses, which serve as vectors to carry the fluorescent protein

fusions into the animal. The fluorescently labeled cells in tissues of interest are

then imaged.

To introduce bioluminescent and fluorescent probes into the animal, a

widely used technique is genetic transfer. The gene that produces

bioluminescence or fluorescence is cloned in the laboratory and introduced into

a laboratory animal in one of two ways. The gene may be inserted into a

harmless virus (called a vector) and introduced into the animal. Or, the gene is

inserted into a stem cell and introduced into the animal so that the differentiated

cell that the stem cell develops into will express the luminescence or fluorescent

protein. The scientists at the forefront of this technology were awarded a Nobel

Prize in Physiology or Medicine.

Adoptive transfer, another technique, involves tagging specific cells,

such as in an animal model of a disease, and transferring those tagged cells

into another laboratory animal to see how they work. Adaptive transfer is used

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to label cells that are “naturally occurring probes” in the body, in that they

migrate to specific targets.

An example is lymphocytes (a family of white blood cells) which are

immune “T” or “B” cells. These immune cells produce antibodies, which are

proteins. Subsets of immune T or B cell lymphocytes are taught by immune

dendritic cells to attack a specific foreign invader, such as an infectious agent,

or a cancer. The newly educated lymphocytes then travel to the site of the

infection or tumor to attack it. The antibodies (proteins) produced by the subset

of T or B cells bind to specific “antigens.” These are surface molecules that are

selectively expressed on specific cell types, including immune cells and also on

tumor cells. Antibodies can be used, therefore, to identify immune cells or

tumors in tissue samples or in live laboratory animals. Scientists label the

antibody protein with a fluorescent tag and inject the material into another

laboratory animal; or, they "stain" tissue sections or isolated cells within tissues

and image the tissues or cells using a technique called flow cytometry.

Use of “Dendrimers” with fluorescent probes is another recent approach

to labeling particles that travel to a target, especially a tumor. Unlike antibodies,

which are proteins produced by living cells, dendrimer nanoparticles are

compounds that are synthesized in the laboratory. They can be made from

various materials (often polymers) that are synthesized and assemble into high

molecular weight spherical particles. Scientists can add to the dendrimers a

fluorescent probe that targets moieties (two divisible parts) and a “payload,”

such as a drug. In this way, dendrimer nanoparticles can be targeted to tumor

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cells and emit light and/or deliver a therapeutic agent that kills the tumors.

Dendrimer nanoparticles can cross the blood brain barrier and enter the brain,

so they are being intensively studied as a potential treatment approach for brain

tumors.

Another technique for imaging and targeting drug delivery is the use of

aptamers, which are made from nucleic acids. Aptamers can be produced to

have exquisite binding specificity for defined molecular targets, similar to

antibodies. They can be attached to the surface polymeric particles for imaging

and to enable targeted drug delivery.

The variations in the ways that different bioluminescent and fluorescent

probes provide information are influenced by the type of microscope or other

imaging technologies used.

Optical Imaging Microscopes

There are three major types of light microscopes: fluorescence, two-

photon and confocal. All three are fluorescent microscopy techniques. Also

widely used are other, older, light microscopy techniques, such as non-

fluorescent bright field microscopy, and various contrast-enhancing techniques

(such as phase contrast, Nomarski, and others).

All three fluorescent light microscopes are used to study molecules in

living cells. They provide insights into how the molecules and the cells they

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compose behave normally, and how they are altered by disease, injury,

treatment, or other experiences. Each type of microscope has its strengths and

limitations.

Fluorescence microscopes are used with fluorescent probes that emit

light of short wavelength to reveal biochemical activities within a cell in human

and animal tissue cultures. Fluorescence microscopes have the highest

resolution of all cellular imaging devices. This enables them to be used to

identify a single fluorescently labeled molecule or differentiate activities of

several differently colored fluorescent molecules in the same cell. This process

is referred to as “subcellular” resolution of molecular activities within a cell.

Fluorescently labeled cells are excited by laser or incandescent light.

The fluorescent label (probe), which is designed to go to a specific molecule or

region of a cell, absorbs a “photon” of energy supplied by the laser or

incandescent light. Then the photon is excited to emit light at a particular

wavelength, depending upon the specific probe used. The emitted light is

recorded as a photographic image, video, fluorescence decay trace, or as

“photo multiplier tube” signals from serial points that are displayed and analyzed

by computer to provide flexible images.

Most fluorescent light microscope probes emit light of short wavelengths,

which is visible with the naked eye, and useful for imaging molecules or cells

close to the surface. Visible light does not pass through tissue well, however,

and these techniques are suitable only in cases where the distances traveled in

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tissue by the probe are small (micrometers in length). As a result, the tissue

grown in laboratory cultures needs to be very thin, from 1 to 20 microns thick, to

be viewed with a fluorescence light microscope. The tissue grown for

visualization with the other two main types of light microscopes, confocal and

multi-photon, can be much thicker.

Confocal laser scanning microscopy provides the ability to

simultaneously collect multiple images in digital form from serial sections of

thick tissue specimens, and flexibly display and analyze them via computer.

Confocal imaging is undertaken in thick tissue cultures and in small laboratory

animals. The blur-free images are taken point by point using “photo multiplier

tubes” that provide sensitive and fast registration of the intensity of emitted light.

The points are then reconstructed by computer, rather than projected through a

microscope’s eyepiece.

Confocal laser scanning imaging relies extensively on fluorescent probes

of longer wavelength to monitor dynamic processes such as: cellular integrity

(differentiating live cells from those that are dying to make way for new cells—

called “apoptosis”—and cell death); membrane fluidity, transduction of cellular

signals, activities of enzymes, and movements of proteins; and, the migration of

cells in the developing animal embryo. Additionally, confocal laser screening

microscopes facilitate study of brain synapses (communication junctions

between two nerve cells) and cell circuitry (formation of neural networks), as do

multi-photon techniques.

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Multi-photon laser microscopy relies on the simultaneous absorption of

two or more photons by a molecule to image fluorescent probes with longer

wavelengths that penetrate deeper into tissues. It is used in thick tissue cultures

and small laboratory animals, often to study cellular actions over time in the

brain. As an example, multi-photon imaging visualizes actions of immune cells

residing in the brain (microglia) and of antibodies summoned to the brain to fight

infections and cancers. Scientists follow the actions of innate immune cells,

called “dendritic” cells, to see how they pass information on how to recognize a

specific invader to certain immune T-cells so that they can attack the intruder.

In addition to studies in human and animal tissues and small laboratory

animals, hand-held fiberoptic confocal and multi-photon devices have been

developed that can be used for endoscopic imaging in humans. This process

entails using a combination of imaging techniques.

Fluorescence resonance energy transfer (FRET) is undertaken with

microscopic imaging to reveal the interaction between two or more fluorescent

probes in tissue cultures. This interaction of adjacent probes is used to monitor

the assembly or fragmentation of molecules, such as occurs in the fusion of two

cell membranes or the binding of a molecule to its receptor. This imaging with

light microscopes is limited to tissue cultures because the signal from

fluorescent-and FRET-generated signals is relatively weak and is poorly

transmitted through living tissue. To undertake such studies in small laboratory

animals requires the use of more sophisticated imaging devices, called

macroscopic optical imaging scanners.

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Macroscopic Optical Imaging in Live Laboratory Animals

Macroscopic Optical scanning techniques image the actions of

molecules and cells that are illuminated with bioluminescent or fluorescent

probes in live laboratory animals. These techniques enable scientists to

visualize actions of cells or molecules anywhere they occur within living small

laboratory animals, and in some cases in laboratory sheep and pigs. Imaging of

live laboratory animals is undertaken in a few major ways.

One approach is to use bioluminescent probes with light microscopes to

image molecules in small laboratory animals, and then to superimpose the

images onto MRI or CT whole body scans of the animal to identify the probes’

anatomical locations. Due to limitations of this bioluminescent imaging

approach, however, the technique of choice for imaging cells and molecules in

laboratory animals is tomographic optical imaging. This technology uses near

infrared (NIR) fluorescent probes.

Optical tomographic imaging of live laboratory animals utilizes

fluorescent probes with near infrared (NIR) light to observe biochemical activity

that occurs deeper within the animals’ tissues. While animal tissue absorbs and

scatters visible light, the amount of NIR light absorbed by tissues is less,

enabling this imaging to penetrate further into laboratory animals’ tissues. In

fact, scientists can detect biochemical cellular activity that occurs hundreds of

micrometers beneath the animals’ skin. The amount of resolution provided by

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NIR alone, however, is insufficient for indentifying which specific cells within any

location are actually emitting the light. This problem is addressed with the

application of genetic and adoptive transfer techniques for creating fluorescent

probes to use with NIR optical imaging. Fluorescence greatly increases the

sensitivity of NIR imaging.

Optical tomography using fluorescent probes and NIR light requires an

additional step compared to imaging whole animals with bioluminescence

probes. In fluorescence optical tomography, light of a specific wavelength must

be shined on the animal. This shined light, in turn, excites the molecule to emit

light at a different wavelength from the light being shined on it. The emitted

light then can be monitored by an imaging device.

Specifically, small animals are placed on a piece of glass in a dark

chamber and the excitation light is switched on. Through the use of filters and a

sensitive electronic camera, light emitted from the fluorescent molecule within

the animal can be separated from the shined light, and quantified. Since the

emitted light comes from many directions, tomographic detectors are placed in

a circle around the animal to collect light coming from various directions.

Computers combine the multiple individual views into three-dimensional

images. Additionally, there have been increasing improvements in resolution,

which now reaches millimeters

Tomographic imaging with fluorescence has two advantages over

bioluminescent whole animal imaging. It allows scientists to increase the

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resolution to more precisely define the point that is the source of the emitted

light. It also allows greater sensitivity and absolute quantification.

While PET imaging also is being used to image small animals in

research, tomographic optical imaging for this purpose is now considered to be

on a par with PET. Unlike PET, however, which is used in human imaging,

tomographic imaging using fluorescently labeled molecules in humans is limited

because the near-infrared light is not able to penetrate as far into thick tissues.

Nonetheless, fluorescent optical tomography is beginning to be attempted in

human breast imaging and colonoscopy, where tissues are not as dense as

other human tissues. NIR probes are also being applied to endoscopy and

surgical guidance in humans.

Combined Imaging Technologies

As these examples illustrate, new combination techniques are already

advancing the threshold of applying imaging innovations to further

understanding brain functions and the effects of experiences, diseases and

therapies in altering these.

A decade ago, we would not envisioned that PET imaging with probes

that attach only to the protein amyloid could help diagnose Alzheimer’s disease

and assess effects of therapies to reduce amyloid or prevent its further

deposition in the brain. We would not have foreseen that PET (which has

relatively poorer spatial resolution) combined with optical imaging in laboratory

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animals could visualize fluorescently marked neurons in the brain to reveal how

one neuron hooks up with another to form neural circuits and to monitor this

process over time during development to see changes in response to disease

or experience.

Since biochemical changes in cells precede changes that occur in

response to disease, identifying these cellular changes could provide the

means to diagnose diseases in their earliest stages, when they are most likely

to be responsive to effective therapies. Imaging biochemical changes in

molecules, rather than physical differences between normal and diseased

tissues, has the potential not only to improve early identification and diagnosis

of diseases, but also to quickly assess the efficacy of various treatments.

Combining molecular imaging with anatomical and physiological imaging

technologies, as these examples illustrate, is fundamentally advancing scientific

understanding of how the brain functions and the translation of that

understanding to improve human health.

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SECTION II

IMAGING TECHNIQUES AT-A-GLANCE

Adoptive transfer is used in molecular imaging to tag specific cells, such as in

an animal model of a disease, and transfer those tagged cells into another

laboratory animal to see how they work. The technique is used to label cells

that are “naturally occurring probes” in the body, such as immune T cells, which

produce antibodies that migrate to an infection. (Please also see Genetic

Transfer, a related technique.)

Angiography uses a radiopaque dye injected through a catheter into a blood

vessel to detect a blockage or narrowing of the vessel. The vessel is outlined on

x-ray as white.

Arterial spin labeling (ASL) is a perfusion contrast used with fMRI. It is used to

quantify regional cerebral blood flow noninvasively to provide absolute

quantification of cerebral blood flow, which renders it sensitive to both static

function and changes occurring over longer intervals.

Bioluminescent probes are used in molecular imaging. They utilize the enzyme

luciferase to generate and emit light by an organism, providing real-time

analyses of disease processes—particularly infections and cancer

progression—at the molecular level in living organisms, including laboratory

36

animals. The enzyme is found in fireflies, glowworms, deep sea marine

organisms and some bacteria and fungi.

BOLD (blood oxygenation level dependent) MRI is the contrast agent used in

most fMRI imaging. (Please see “Functional MRI” below.) BOLD contrast

reflects a complex interaction between the volume of blood, its flow, and its

transport of oxygen by an iron-containing protein in red blood cells. Functional

contrast is produced when the oxygen is released from the iron and taken up

and used by brain cells (indicating that they are active). After the iron looses the

oxygen, the iron becomes highly magnetized when exposed to the MRI

magnetic field.

Computer Assisted Tomography (CT) uses special x-ray equipment to obtain

three-dimensional anatomical images of bone, soft tissues and air. An x-ray

emitter rotated around the head measures the rays’ intensities from different

angles. Sensors measure the amount of radiation absorbed by different tissues;

a computer uses the differences in X-ray absorption to form cross-sectional

images or “slices” of brain called “tomograms.” CT can be done quickly, and so

is used extensively in the ER to identify evidence of brain trauma, such as

swelling or bleeding (as from hemorrhagic stroke or a ruptured brain aneurysm).

Confocal laser scanning microscopy is a molecular imaging technique that

monitors dynamic processes such as synaptic activity and cell death. The

technique enables simultaneous collection in digital form of multiple images

from serial sections of thick tissue specimens that are flexibly displayed and

37

analyzed via computer. The blur-free images are taken point by point, and with

sensitive and fast registration of the intensity of emitted light, are reconstructed

via computer.

Deep brain stimulation (DBS) involves implanting electrodes in specific areas in

the brain and externally stimulating the electrodes to measure electrical

activities of neurons and their electrochemical pathways. DBS is used

therapeutically to treat intractable Parkinson’s disease and essential tremor,

and is being studied for possible use in intractable depression and other brain

conditions. It is also used in a few highly specialized centers to explore the

neuronal underpinnings of cognition.

Dendrimer Nanoparticles are used in molecular imaging with fluorescent probes

to label particles that travel to a target, especially a tumor. Dendrimer

nanoparticles are compounds that are synthesized in the laboratory from

materials (often polymers) and assembled into high molecular weight spherical

particles. A fluorescent probe is added to the dendrimer nanoparticles which

then target and light up specific cells. A therapeutic drug also can be added to

dendrimer nanoparticles, which can cross the blood brain barrier and enter the

brain. They are being intensively studied, therefore, as a means to deliver

potential treatment targeted to brain tumor cells.

Diffusion-Perfusion-weighted MRI is a combination technique used to estimate

the “ischemic penumbra.” This is the brain tissue that has suffered from

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reduced blood flow following ischemic stroke but has not yet died and is the

target of intensive therapy.

Diffusion-tensor MRI (DTMRI) measures microscopic water motion in tissues,

and in the brain this motion is facilitated along white matter tracts that connect

brain regions. Computerized mathematical models construct the images of the

white matter tracts. It is used extensively pre-surgically to plan, such as to

identify and spare these tracts during surgical removal of a brain tumor.

Diffusion-weighted MRI shows whether brain tissue has been damaged due to

insufficient blood flow to the tissue.

Electroencephalography (EEG) measures the electrical activity that is produced

by neurons as recorded from electrodes placed along the scalp.

Fluorescence resonance energy transfer (FRET) is a molecular imaging

technique that reveals the interaction between two or more fluorescent probes

in tissue cultures. It is used, for instance, to visualize a molecule binding to its

receptor on a cell.

Functional MRI (fMRI) shows the brain in action. It is a highly sensitive but

indirect measure that is used to elucidate processes involved in higher cognitive

functioning, including identification of motor and task activation areas; and

reorganization of function following injury to a single brain area. It is based on

the principle that changes in regional cerebral blood flow and metabolism are

coupled to changes in regional neural activity involved in brain functioning, such

39

as memorizing a phrase or remembering a name. Almost all fMRI techniques

use the contrast mechanism called BOLD (please see BOLD above).

Fluorescence microscopes are used with fluorescent probes that emit light of

short wavelength to reveal biochemical activities within a cell in human and

animal tissue cultures. These microscopes have the highest resolution of all

cellular imaging devices. They can be used to identify a single fluorescently

labeled molecule or differentiate activities of several differently colored

fluorescent molecules in the same cell.

Fluorescent probes are used in molecular imaging to visualize molecules and

their actions. The probes are green fluorescent protein, its yellow, blue and

cyan-colored mutants, and red fluorescent proteins. Fluorescent probes that

emit light of short wavelengths are used with fluorescent light microscopes to

image molecules that are close to the surface in laboratory cultures of thin

human or animal tissues. Fluorescent probes that emit light of longer

wavelengths are introduced into small laboratory animals and used with other

microscopes (please see Confocal laser scanning microscopy and Multi-photon

laser microscopy) and excited by ultraviolet light to show molecules deeper in

tissues, in the small laboratory animal or in thick animal tissues in laboratory

cultures.

Intravital Light Microscopic technologies use light-emitting probes as contrast

to visualize the activities of specific molecules and the cells they compose.

Imaging is undertaken in tissues surgically biopsied from humans and

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laboratory animals. Imaging is also undertaken in small laboratory animals, but

is confined to the specific location under view.

Genetic Transfer is used in molecular imaging to introduce bioluminescent and

fluorescent probes into the animal. The gene that produces bioluminescence or

fluorescence is cloned in the laboratory and introduced into a laboratory animal.

The gene is introduced into the laboratory animal either by inserting it into a

harmless virus (called a vector) that gets into a specific type of cell, or by

inserting it into a stem cell that differentiates into a cell that expresses the

luminescence or fluorescent protein. (Please also see Adoptive Transfer, a

related technique.)

Intravital Macroscopic Imaging technologies use light-emitting probes as

contrast to visualize specific molecules and the cells they compose in small

laboratory animals and in a few larger laboratory animals. The molecules can

be imaged everywhere they occur in body as opposed to a single location

(please see intravital light microscope technologies); and, the molecules can be

imaged as they move throughout the body, including the brain. (Please also

see Macroscopic Optical Scanning techniques.)

Laser Doppler Ultrasound employs laser technology to combine information

from both light and sound. It is a non-invasive and highly sensitive method for

measuring even tiny changes in the rate of blood flow velocity (speed) within

arteries throughout the body, including the brain. Its primary use in the brain is

41

for monitoring severely head injured patients, especially those in coma, in

intensive care units.

Macroscopic Optical Scanning techniques image the actions of molecules and

cells that are illuminated with bioluminescent or fluorescent probes in live

laboratory animals. These techniques enable scientists to visualize actions of

cells or molecules anywhere they occur within living small laboratory animals,

and in some cases in laboratory sheep and pigs. (Please also see Intravital

Macroscopic Imaging Technologies.)

Magnetoencephalography (MEG) maps brain activity by measuring magnetic

fields that are generated by neural activity in the brain. It is used to investigate

the basis of sensory processing and motor planning in the brain.

Magnetic Resonance Imaging (MRI) is a non-invasive technology with high

resolution that is used primarily to image brain structure and function. It is

based on the principle that changes in regional cerebral blood flow and

metabolism are coupled to changes in regional neural activity involved in brain

functioning. Significant contrast in tissue can be attributed to changes either in

blood flow alone, or in metabolism alone, or in blood flow and metabolism.

(Please also see Structural MRI.)

Magnetic Resonance Spectroscopy (MRS) is a non-invasive technique that

measures biochemical changes in the brain over time, characterizing brain

diseases according to the natural history of the chemical changes produced.

MRS is conducted in an MRI scanner, uses magnetization and radio waves

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from hydrogen protons in non-water atoms, such as carbon and nitrogen, and

produces a color chart (“spectra”) that reflects the concentrations of molecules

according to their chemical composition.

Multi-photon laser microscopy is a molecular imaging technology that is used to

study the actions of specific cells in the brain over time. The technology relies

on the simultaneous absorption of two or more photons by a molecule to image

fluorescent probes with long wavelengths that penetrate deep into tissues. It is

used in thick tissue cultures and small laboratory animals.

Optical Probes are used in cellular and molecular imaging. They are molecules

that have been specially labeled to emit light of various wavelengths, to

“contrast” the target cells of interest from other cells.

Optical tomographic imaging is a molecular imaging technology used to study

biochemical activity that occurs deep within the tissues of live laboratory

animals. Near infrared (NIR) light is used in combination with fluorescent

probes. Light of a specific wavelength is shined on the animal; in turn this light

excites the target molecule to emit light at a different wavelength, which is

monitored by tomographic detectors placed in a circle around the animal to

collect light coming from various directions. Computers combine the multiple

individual views into three-dimensional images.

Perfusion-weighted MRI shows areas of the brain in which blood flow has been

altered.

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Positron Emission Tomography (PET) measures physiological functioning in the

brain. It provided the first opportunity to explore the parts of the brain that were

activated in undertaking specific tasks; now it is primarily used to study

neurotransmitters, actions of pharmaceutical drugs, and the expression of

specific genes in the brain. PET is based on the principle that changes in

regional cerebral flow and metabolism in brain regions are coupled to changes

in neural activity in those regions. PET uses ionizing radiation (radioisotopes)

as tracers. Each radioisotope attaches to a specific molecule (carbon, nitrogen,

oxygen and fluorine). The regional distribution of exogenously administered

positron-emitting tracers is measured using tomographic imaging. PET can

quantify tiny concentrations of the radioisotope tracer so its measurements of

change are exquisitely sensitive. PET, using new tracers that attach solely to

the protein beta amyloid, may become a means to help diagnose Alzheimer’s

disease and identify patterns predictive of conversion from mild cognitive

impairment to Alzheimer’s.

Structural MRI measures the nuclear magnetic resonance of the body’s own

molecules, water protons, to create a computerized three-dimensional image of

tissues. Variations in water located in different brain structures and

compartments provide contrast, and the ability to see the spatial orientation of

various brain structures. The contrast differentiates the brain’s gray matter

(primarily nerve cell bodies) from white matter (primarily axons and their myelin

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sheaths) which are the nerve cell communication cables that connect brain

regions. Many disease processes result in water content changes; these are

reflected in the image produced to provide diagnostic information.

Single Photon Emission Computed Tomography (SPECT) measures

physiological functioning in the brain and is similar to PET (Please see Positron

Emission Tomography). In contrast to PET, SPECT uses commercially

available stable low level radioisotopes and is therefore less expensive, more

convenient for clinical use, is widely used clinically.

Transcranial magnetic stimulation (TMS) is a non-invasive technique that is

used to map cortical functions in the brain, such as identifying motor or speech

areas. With TMS, a large electromagnetic coil is placed on the scalp, near the

forehead. An electromagnet is then used to create a rapidly changing magnetic

field, inducing weak electric currents. Unlike the mapping function, a repetitive

form of TMS, called rTMS, is used therapeutically to treat depression.

Ultrasound uses sound waves to determine the locations of surfaces within

tissues, and differentiates surfaces from fluids. It does so by measuring the time

that occurs between the production of an ultrasonic pulse to the production of

the echo created when the surface reflects the pulse.

X-rays measure the density of tissues. They use photons, a quantum of visible

light that possesses energy. The photons are passed through the body,

deflected and absorbed to different degrees by tissues, and recorded as they

pass out of the body onto a silver halide film. Dense structures such as bone,

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which block most of the photons, appear white; structures containing air appear

black; and muscle, fat and fluids appear in various shades of gray.

November, 2011